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Bacteriophage T4 lysozyme is a basic molecule with an isoelectric point above
9.0, and an excess of nine positive charges at neutral pH. It might be expected
that it would be energetically costly to bring these out-of-balance charges from
the extended, unfolded, form of the protein into the compact folded state. To
determine the contribution of such long-range electrostatic interactions to the
stability of the protein, five positively charged surface residues, Lys16,
Arg119, Lys135, Lys147 and Arg154, were individually replaced with glutamic
acid. Eight selected double, triple and quadruple mutants were also constructed
so as to sequentially reduce the out-of-balance formal charge on the molecule
from +9 to +1 units. Each of the five single variant proteins was crystallized
and high-resolution X-ray analysis confirmed that each mutant structure was, in
general, very similar to the wild-type. In the case of R154E, however, the
Arg154 to Glu replacement caused a rearrangement in which Asp127 replaced Glu128
as the capping residue of a nearby alpha-helix. The thermal stabilities of all
13 variant proteins were found to be fairly similar, ranging from 0.5 kcal/mol
more stable than wild-type to 1.7 kcal/mol less stable than wild-type. In the
case of the five single charge-change variants, for which the structures were
determined, the changes in stability can be rationalized in terms of changes in
local interactions at the site of the replacement. There is no evidence that the
reduction in the out-of-balance charge on the molecule increases the stability
of the folded relative to the unfolded form, either at pH 2.8 or at pH 5.3. This
indicates that long-range electrostatic interactions between the substituted
amino acid residues and other charged groups on the surface of the molecule are
weak or non-existent. Furthermore, the relative stabilities of the multiple
charge replacement mutant proteins were found to be almost exactly equal to the
sums of the relative stabilities of the constituent single mutant proteins. This
also clearly indicates that the electrostatic interactions between the replaced
charges are negligibly small. The activities of the charge-change mutant
lysozymes, as measured by the rate of hydrolysis of cell wall suspensions, are
essentially equal to that of the wild-type lysozyme, but on a lysoplate assay
the mutant enzymes appear to have higher activity.(ABSTRACT TRUNCATED AT 400
WORDS)
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